Antithrombin III, a heparin cofactor, is the major plasma inhibitor of the coagulation proteases. Alone, antithrombin III reacts slowly with these enzymes to form a 1:1 inactive complex. Addition of heparin, however, greatly enhances the rate of protease inactivation without altering the stoichiometry of the reaction. This increase in reactivity appears to result from heparin induced conformational changes in antithrombin III and the coagulation proteases. We have shown that chemical modification of a single tryptophan residue in antithrombin III blocks heparin binding and the heparin-promoted enhancement of thrombin inactivation. Using a variety of tryptophan reagents we will compare the effects of chemical modification on the functional properties of human, bovine and canine antithrombin III. We will also use this specifically labeled tryptophan as an aid to the isolation and amino acid sequence analysis of the heparin binding sites of these three antithrombins. This will allow us to determine sequence homologies and differences in this site. To further define the structural requirements for heparin binding we propose to use limited proteolytic digestion of native antithrombin III for the production of functional fragments containing the heparin binding domain. These approaches should enable us to identify the heparin binding site within the antithrombin III molecule and its relationship to the site of protease interaction.